Mucin-type O-glycosylation, one of the most abundant forms of protein glycosylation, is found on secreted and cell surface associated glycoproteins of all eukaryotic cells except yeast. Mucin-type O-glycans contribute to a number of important molecular functions, including: direct effects on protein conformation, solubility, and stability; specific receptor functions that regulate cell trafficking and cell-cell interactions; and microbial clearance. Mucin-type O-glycans are synthesised in the Golgi through the sequential addition of saccharide residues, first to hydroxyl groups on serines and threonines of a protein core and subsequently to hydroxyl groups on the growing saccharide chains that extend from the protein core. There is great diversity in the structures created by O-glycosylation (hundreds of potential structures), which are produced by the catalytic activity of hundreds of glycosyltransferase enzymes that are resident in the Golgi complex. Diversity exists at the level of the glycan structure and in positions of attachment of O-glycans to protein backbones. Despite the high degree of potential diversity, it is clear that O-glycosylation is a highly regulated process that shows a high degree of conservation among multicellular organisms.
The factors that regulate the attachment of O-glycans to particular protein sites and their extension into specific structures are poorly understood. Longstanding hypotheses in this area propose that mucin-type O-glycosylation occurs in a stochastic manner where structure of acceptor proteins combined with topology and kinetic properties of resident Golgi glycosyltransferases determine the order and degree of glycosylation (1). This concept does not fully explain the high degree of regulation and specialisation that governs the O-glycosylation process. In particular it is difficult to envision how large mucin molecules with high densities of O-glycans are glycosylated in the Golgi by stochastic mechanisms that also create other sparsely glycosylated proteins.
The first step in mucin-type O-glycosylation is catalysed by one or more members of a large family of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases) (EC 2.4.1.41), which transfer GalNAc to serine and threonine acceptor sites42. To date twelve members of the mammalian GalNAc-transferase family have been identified and characterized, and several additional putative members of this gene family have been predicted from analysis of genome databases. The GalNAc-transferase isoforms have different kinetic properties and show differential expression patterns temporally and spatially, suggesting that they have distinct biological functions42. Sequence analysis of GalNAc-transferases have led to the hypothesis that these enzymes contain two distinct subunits: a central catalytic unit, and a C-terminal unit with sequence similarity to the plant lectin ricin, designated the “lectin domain” (3-6). Previous experiments involving site-specific mutagenesis of selected conserved residues confirmed that mutations in the catalytic domain eliminated catalytic activity. In contrast, mutations in the “lectin domain” had no significant effects on catalytic activity of the GalNAc-transferase isoform, GalNAc-T1 (3). Thus, the C-terminal “lectin domain” was believed not to be functional and not to play roles for the enzymatic functions of GalNAc-transferases (3).
Recent evidence demonstrates that some GalNAc-transferases exhibit unique activities with partially GalNAc-glycosylated glycopeptides. The catalytic actions of at least three GalNAc-transferase isoforms, GalNAc-T4, -T7, and -T10, selectively act on glycopeptides corresponding to mucin tandem repeat domains where only some of the clustered potential glycosylation sites have been GalNAc glycosylated by other GalNAc-transferases7-9, 44. GalNAc-T4 and -T7 recognize different GalNAc-glycosylated peptides and catalyse transfer of GalNAc to acceptor substrate sites in addition to those that were previously utilized. One of the functions of such GalNAc-transferase activities is predicted to represent a control step of the density of O-glycan occupancy in mucins and mucin-like glycoproteins with high density of O-glycosylation. It was hypothesized that such sequential actions of multiple GalNAc-transferase isoforms may be required to complete O-glycan attachments to some mucin peptide sequences allowing for detailed control of density.
One example of this is the glycosylation of the cancer-associated mucin MUC1. MUC1 contains a tandem repeat O-glycosylated region of 20 residues (HGVTSAPDTRPAPGSTAPPA) (SEQ ID NO: 1) with five potential O-glycosylation sites. GalNAc-T1, -T2, and -T3 can initiate glycosylation of the MUC1 tandem repeat and incorporate at only three sites (HGVTSAPDTRPAPGSTAPPA (SEQ ID NO: 1), GalNAc attachment sites underlined). GalNAc-T4 is unique in that it is the only GalNAc-transferase isoform identified so far that can complete the O-glycan attachment to all five acceptor sites in the 20 amino acid tandem repeat sequence of the breast cancer associated mucin, MUC1. GalNAc-T4 transfers GalNAc to at least two sites not used by other GalNAc-transferase isoforms on the GalNAc4TAP24 glycopeptide (TAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID NO: 2), GalNAc attachment sites underlined) (8). An activity such as that exhibited by GalNAc-T4 appears to be required for production of the glycoform of MUC1 expressed by cancer cells where all potential sites are glycosylated (10). Normal MUC1 from lactating mammary glands has approximately 2.6 O-glycans per repeat (11) and MUC1 derived from the cancer cell line T47D has 4.8 O-glycans per repeat (10). The cancer-associated form of MUC1 is therefore associated with higher density of O-glycan occupancy and this is accomplished by a GalNAc-transferase activity identical to or similar to that of GalNAc-T4.
The specific mechanism by which GalNAc-T4, -T7, and -T10 recognize and function with GalNAc-glycosylated glycopeptides is not known. However, it was originally demonstrated that the GalNAc-glycopeptide specificity exerted by GalNAc-T4 is directed or at least dependent on its lectin domain. A single amino acid substitution in the T4 lectin domain predicted to inactivate its function abolished the GalNAc-glycopeptide specificity of T4 without adversely affecting the basic catalytic mechanism of the transferase42. This suggests that the lectin domain interacts with GalNAc-glycopeptides and confers a novel catalytic function to the enzyme protein. Despite extensive attempts it has in the past not been possible to demonstrate actual binding of the transferase and lectin to sugars and glycopeptides, but it was possible to demonstrate selective inhibition of the GalNAc-glycopeptide activity of GalNAc-T4 using 230 mM concentration of GalNAc42. Millimolar concentrations of GalNAcα-benzyl can inhibit the lectin mediated GalNAc-glycopeptide substrate specificity of GalNAc-T4 as well as -T7. Polypeptide GalNAc-transferases, which have not displayed apparent GalNAc-glycopeptide specificities, also appear to be modulated by their lectin domains. Recently, it was found that mutations in the GalNAc-T1 lectin domain, similarly to those previously analysed in GalNAc-T442, modified the activity of the enzyme in a similar fashion as GalNAc-T4. Thus, while wild type GalNAc-T1 added multiple consequtive GalNAc residues to a peptide substrate with multiple acceptor sites, mutated GalNAc-T1 failed to add more than one GalNAc residue to the same substrate45. The mechanism is however not understood.
Glycosylation confers physico-chemical properties including protease resistance, solubility, and stability to proteins (12-14). Glycosylation furthermore confers changes in immunological responses to proteins and glycoproteins. O-glycosylation on mucins and mucin-like glycoproteins protect these molecules found in the extracellular space and body fluids from degradation. Control of O-glycosylation with respect to sites and number (density) of O-glycan attachments to proteins as well as control of the O-glycan structures made at specific sites or in general on glycoproteins, is of interest for several purposes. Diseased cells e.g. cancer cells often dramatically change their O-glycosylation and the altered glycans and glycoproteins may constitute targets for therapeutic and diagnostic measures (15; 16). Mucins functioning in body fluids may have different properties depending on density and structure of O-glycans attached in protection against disease, including infections by micro-organisms. Furthermore, mucins with different glycosylation may change physico-chemical properties including stability and solubility properties that may influence turnover and removal of mucous. A number of lung diseases, e.g. cystic fibrosis, asthma, chronic bronchitis, smokers lungs, are associated with symptomatic mucous accumulation (17-19), and it is likely that the nature and structure of mucins play a role in the pathogenesis of such diseases.
Partial inhibitors of O-glycosylation in cells have been reported. Aryl-N-acetyl-α-galactosaminides such as benzyl-, phenyl-, and p-nitrophenyl-GalNAc were originally found to inhibit the second step in O-glycosylation, the O-glycan processing step, by inhibiting synthesis of core 1 (Galβ1-3GalNAcα1-R) and more complex structures 20. Benzyl-αGalNAc was also found to inhibit sialylation. It is generally believed that the downstream effects of benzyl-αGalNAc treatment are mediated by substrate competition of biosynthetic glycosylation products of benzyl-αGalNAc. Thus, e.g. the immediate glycosylation product of benzyl-αGalNAc is Galβ1-3GalNAcα-benzyl and this serves as an efficient substrate for the core 1 α2-3sialyltransferase ST3Gal-I21,22. GalNAcα-benzyl has been the most widely used inhibitor of O-glycosylation, but it has only been used in cell culture as effective treatment concentrations lead to intracellular build-up of vesicles with GalNAcα-benzyl products and treated cells change morphology and growth characteristics46.
Treatment of cells with benzyl-αGalNAc inhibit O-glycan processing and affect apical sorting of some O-glycosylated proteins23-25. The mechanism for this is generally believed to be through inhibition of sialylation46. Inhibition of mucin secretion has also been observed in culture cells, more specifically HT29 MTX cells, but this effect is not generally found in mucin secreting cells46.
True inhibitors of O-glycosylation, i.e. inhibitors of the initial O-glycan attachment process governed by polypeptide GalNAc-transferases have not been identified.
Inhibitors of the initiating step in O-glycosylation could completely or selectively block attachment of O-glycans to O-glycosylation sites in proteins. Compounds inhibiting the catalytic function of a selected subset of the polypeptide GalNAc-transferase family may be predicted to only lead to partial inhibition of O-glycosylation capacity of cells. Proteins with no or little O-glycosylation may have entirely different biological properties than their normal glycosylated counterparts. Complete inhibition of O-glycosylation is not desirable because of the many diverse functions of O-glycans, and it is expected to result in cell death. Selective inhibition of O-glycosylation on the other hand is desirable in many cases such as cancer cells producing glycoproteins and mucins with more dense O-glycosylation than normal cells. For example breast cancer cells appear to hyperglycosylate the cancer-associated cell surface mucin MUC1 compared to glycosylation in normal cells (10). The overexpression of MUC1 and hyperglycosylation found in cancer cells are likely to be important for the pathobiology of cancers. Methods of inhibiting the hyperglycosylation of mucins in cancer cells is desirable.
It is apparent from the above that inhibitors in the prior art interfere with O-glycan processing, i.e. the glycosylation process that extend GalNAc residues directly attached to proteins at serine and threonine residues. Existing inhibitors of O-glycosylation are not suitable for therapeutic treatment in mammals including man as they profoundly affect O-glycosylation processing as well as lead to undesired morphological and growth effects on culture cells.
Consequently, there exists a need in the art for methods of inhibiting the functions of polypeptide GalNAc-transferases. Preferable in selectively inhibiting O-glycosylation attachments in glycoproteins and mucins. There also exists a need in the art for therapeutic compounds that display selectively and limited inhibition of O-glycosylation without generally affecting the process of O-glycosylation. The present invention meets these needs, and further presents other related advantages.